The ability to discern mutations is important in many ways, but is especially important for cancer research and clinical diagnostics. Certain polymorphic variations in nucleic acid sequences, commonly as small as single nucleotide polymorphism (SNP), serve as markers of cancer development and progression. Identification and detection of these sequence variations enables early cancer detection from tissue biopsies and body fluids such as plasma or serum; assessment of residual disease after radio- and chemotherapy; disease staging and molecular profiling for prognosis or tailoring therapy to individual patients; and monitoring of the therapy outcome and cancer remission. The first problem of cancer diagnostics is that the DNA or RNA prognostic markers are usually present in very limited amounts, commonly less than <1,000 molecules per sample. Quantitative and accurate detection of these marker loads remains challenging even though, in theory, certain DNA amplification protocols including Polymerase Chain Reaction (PCR) can quickly increase the target concentration to a detectable level from as little as a single DNA molecule. The main challenge of cancer diagnostics, however, is that the clinical samples are typically composed of both normal (wild-type) and mutant DNAs, and the quantity of normal DNA often vastly exceeds the mutant loads, making it very difficult to detect and identify the minority alleles. This means that, in order to be used as a routing tool, any particular method in cancer diagnostics has to be both highly sensitive and exceptionally sequence-specific. An ideal assay should be fast (real-time), cost-effective, simple to use, and capable of detecting limited amounts of mutant alleles (1-100 copies) in the presence of 105-106 times excess of normal gene sequences. More than a dozen of different methods have been developed and tested to date (Gocke C. D. et al, 2000; Milbury C. A. et al, 2009; Parsons B. L., Heflich R. H., 1997; Zhou L. et al., 2010).
Despite the considerable progress achieved, the main problem has not yet been solved and the ideal mutant-enriching remains to be developed. The majority of the developed methods exhibit insufficient mutant-enrichment power (102-103). In cases where excellent mutant-enrichment is achieved, a particular assay is either too complex or multistage like APRIL-ATM (Kaur M. et al, 2002) and difficult to automate. The bi-PAP-ASA technology (Liu Q., Sommer S. S., 2000; Shi J. et al, 2007) provides limited capabilities to adapt probe-based detection. The RFLP-PCR assay family (Parry J. M. et al, 1990; Jenkins G. J. S. et al, 1998) is very powerful in mutant enrichment but these technologies are generally limited to the mutations located in restriction nucleases' sites while all other sequences are not covered. There is therefore a pronounced need in the art for more efficient and versatile methods of nucleic acids detection that can provide an exceptionally high level of mutant allele enrichment, up to 106 or more, and identify the target sequence variation as small as SNP regardless of the polymorphism, its target location and surrounding sequence contents.